Somatic stem cells

ABSTRACT

A somatic stem cell that is CD10+, CXCR4+, and CD31+ and another somatic stem cell that is CD105+, CD44+, and nestin+. Also disclosed are both a method of preparing these stem cells and a method of using them to treat degenerative diseases, e.g., a muscle-degenerative disease. The invention further includes making and using liver cells derived from the somatic cell that is CD105+, CD44+, and nestin+.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a division of U.S. application Ser. No. 14/877,585,filed on Oct. 7, 2015, which is a division of U.S. application Ser. No.13/629,884, filed on Sep. 28, 2012, which claims priority to U.S.Provisional Application No. 61/540,191, filed on Sep. 28, 2011, and alsoto U.S. Provisional Application No. 61/609,522, filed on Mar. 12, 2012.The contents of all prior applications are hereby incorporated byreference herein in their entirety.

BACKGROUND

Stem cells are totipotent, pluripotent, or multipotent cells that candifferentiate in vivo or in vitro into many or all cell types. Due totheir pluripotency, embryonic stem (ES) cells are believed to hold greatpotential for treating degenerative or inherited diseases. Yet, ethicalconsiderations have hampered the use of human ES cells. Stem cells of anon-embryonic origin would circumvent this obstacle. Thus, there is aneed for non-embryonic stem cells.

SUMMARY

This invention relates to somatic stem cells and related methods. Oneaspect of this invention relates to an isolated somatic non-adherentstem cell that is CD10+, CXCR4+, and CD31+. This cell is named “SB-3cell” herein. The sign “+” following a cell marker stands for a higherfluorescent staining with a marker-specific antibody, as compared to alower fluorescent staining with an isotype control of the antibody. Thesign “−” following a cell marker stands for the same fluorescentstaining with a marker-specific antibody as that with an isotype controlof the antibody.

Another aspect of this invention relates to an isolated somatic adherentstem cell that is CD105+, CD44+, and nestin+. This cell is named “SB-4cell” herein.

In still another aspect, the invention features a cell bank including aplurality of populations of somatic stem cells. The somatic stem cellsare each CD10+, CXCR4+, and CD31+, and the populations are originatedfrom different subjects. The subjects can be humans or non-humans.

In yet another aspect, the invention features a cell bank including aplurality of populations of somatic stem cells. The somatic stem cellsare each CD105+, CD44+, and nestin+, and the populations are originatedfrom different subjects. The subjects can be humans or non-humans.

Within the scope of this invention as well is a method for treating amuscle injury or a muscle-degenerative disease. The method includesadministering to a subject in need thereof an effective amount of thesomatic stem cells that are CD10+, CXCR4+, and CD31+. Examples of themuscle-degenerative disease include muscular dystrophy, fibromyalgia,myopathies, dermatomyositis, polymyositis, rhabdomyolysis, andmyocarditis.

The invention further features another method for treating a muscleinjury or a muscle-degenerative disease. The method includesadministering to a subject in need thereof an effective amount of thesomatic stem cells that are CD105+, CD44+, and nestin+.

Also contemplated herein is a method of preparing somatic stem cells.The method includes (1) obtaining from a subject (e.g., a human ornon-human) a bodily fluid sample (e.g., blood, bone marrow, umbilicalcord blood, menstrual fluid, and amniotic fluid) containing a pluralityof cells, (2) incubating the sample with a divalent cation chelatingagent (e.g., EDTA, EGTA, and sodium citrate) or heparin in a containeruntil the sample is separated into an upper layer and a lower layer, (3)collecting the upper layer, (4) isolating from the upper layer apopulation of somatic stem cells that are 0.3-6.0 micrometers in size,and (5) culturing the isolated somatic stem cells in a medium containingspecific growth factors: R-Spondin-1, SCF, G-CSF, bFGF, EGF, and PDGF.Somatic stem cells can be positive for CD10, CXCR4, and CD31. They canalso be positive for CD105, CD44, and nestin.

Further contemplated is a method of preparing liver cells from theabove-described somatic stem cells that are CD105+, CD44+, and nestin+.The method includes culturing the isolated somatic stem cells in a firstdifferentiating medium containing activin; culturing the isolatedsomatic stem cells in a second differentiating medium containing basicfibroblast growth factors (bFGF) and bone morphogenetic protein 2(BMP2); further culturing the isolated somatic stem cells in a thirddifferentiating medium containing hepatocyte growth factor (HGF),dexamethasone (DEX), and oncostatin M (OSM); and collecting liver cellsthus obtained, the liver cells expressing albumin, transferrin, andhepatocyte nuclear factor 3B (HNF3B).

The invention also includes an extracorporeal bioartificial liverdevice. The device has a cartridge that contains an array of hollowfibers and the liver cells prepared in the manner described above. Theliver cells express one or more proteins selected from the groupconsisting of albumin, alpha-1-antitrypsin, factor V, complement C3,antithrombin III, and transferrin. They are placed in the extracapillaryspace between the hollow fibers, which are each formed of a membranehaving a pore size of about 0.1 μm to 0.3 μm. The cartridge can have acylindrical shape with a first opening on the side wall close to oneterminus and a second opening also on the side wall but close to theother terminus. The first opening is affixed to a first passage and thesecond opening is affixed to a second passage, the two passages eachextending away from the cartridge.

In addition, the invention features a method of treating acute liverfailure. The method includes identifying a subject in need of treatment,attaching the above-described extracorporeal bioartificial liver deviceto an artery of the subject through the first passage and a vein of thesubject through the second passage, perfusing blood from the subjectthrough the capillary space inside each of the hollow fibers in thecartridge, and allowing cleansing of blood by permitting the crossoverof toxic solutes from the blood to the liver cells cultured in theextracapillary space between the hollow fibers and also allowing thediffusion of vital metabolites from the liver cells to the bloodreturning to the subject. The subject can be a human who suffers from achronic liver disease, which most commonly results from hepatitis Cinfection, alcohol abuse, and drug overdose. In an embodiment, thesubject is awaiting liver transplantation.

Also described herein is a method of producing albumin. The methodincludes culturing in a medium liver cells prepared using the methoddescribed above, and collecting from the medium albumin produced by theliver cells.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawing, and from the claims.

DESCRIPTION OF DRAWING

FIG. 1 is a photograph showing SB-3 cells and SB-4 cells in a culture.

FIG. 2 is a schematic presentation of an extracorporeal bioartificialliver device that is attached to a human subject.

FIG. 3a is a photograph of an exemplary cartridge that can be used in anextracorporeal bioartificial liver device.

FIG. 3b is an image of a cross-section of the exemplary cartridge.

DETAILED DESCRIPTION

This invention is based, at least in part, on the unexpected discoveriesthat (i) non-adherent cells that are positive for CD10, CXCR4, and CD31(i.e., SB-3 cell) can be differentiated into three germ layers; and (ii)that these cells can become adherent cells that are positive for CD105,CD44, and nestin (i.e., SB-4 cell).

SB-3 Cells and SB-4 Cells

SB-3 and SB-4 cells can be prepared from a cell population that isisolated from a bodily fluid sample (e.g., blood, bone marrow, umbilicalcord blood, menstrual fluid, and amniotic fluid). The bodily fluidsample can be obtained from a human or from a non-human. Examples of anon-human, from which the above-mentioned somatic stem cells can beobtained, include, but are not limited to primate, dog, rodent, guineapigs, cat, horse, cow, sheep, and pig. Indeed, pet animals, farmanimals, experimental animals, and disease-model animals are allcontemplated.

A bodily fluid is drawn from a subject and incubated with a divalentcation chelating agent (e.g., EDTA, EGTA, and sodium citrate) or heparinin a container until the sample is separated into an upper layer and alower layer. From the upper layer, a population of somatic stem cellsthat are 0.3-6.0 μm in size are isolated and then cultured in a mediumcontaining certain growth factors (i.e., R-Spondin-1, SCF, G-CSF, bFGF,EGF, and PDGF) for 1 to 30 days (e.g., 4 to 14 days). The medium cancontain each at a concentration of 1 to 100 ng/ml (e.g., 2 to 50 ng/mland 5 to 20 ng/ml). Under these culturing conditions, the diameters ofthe cells increase to 6-25 μm.

The cells that remain non-adherent are SB-3 cells and those that becomeadherent are SB-4 cells. The somatic stem cells in the SB cellpopulation can be CD9+, SSEA1+, SSEA4+, CD13+, or Strol+. For example,some are CD9+CD349+.

Both SB-3 and SB-4 cells are somatic stem cells. They can be used toregenerate differentiated and functional cells for treating variousdegenerative disorders or tissue damage. These cells can be easilymaintained and expanded in vitro, and induced to differentiation usingroutine technical approaches. In addition, after grafting these stemcells into an animal subject (e.g., a mouse), there is no evidence ofmalignant growth. These stem cells contain a normal chromosomalcomplement. They are responsive to lineage-induction agents,proliferation agents, and differentiation inhibitory agents. Due tothese advantages, they represent an alternative to other stem cells.

The term “stem cell” herein refers to a cell that is totipotent,pluripotent, multipotent, or unipotent, i.e., capable of differentiatinginto one or more terminally differentiated cell types. Totipotent stemcells typically have the capacity of developing into any cell type. Theycan be both embryonic and non-embryonic in origin.

Pluripotent cells are typically capable of differentiating intoectoderm, endoderm, and mesoderm cells. Multipotent cells candifferentiate into several different, terminally differentiated celltypes. Unipotent stem cells can differentiate into only one cell type.They have the property of self-renewal, which distinguishes them fromnon-stem cells. The above-mentioned stem cells can originate fromvarious tissues or organs, including, but are not limited to, blood,nerve, muscle, skin, gut, bone, kidney, liver, pancreas, and thymus.

The stem cells disclosed herein are substantially pure. The term“substantially pure”, when used in reference to stem cells or cellsderived therefrom (e.g., differentiated cells), means that the specifiedcells constitute the majority of cells in the preparation (i.e., morethan 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%). Generally, asubstantially purified population of cells constitutes at least about70% of the cells in a preparation, usually about 80% of the cells in apreparation, and particularly at least about 90% of the cells in apreparation (e.g., 95%, 97%, 99% or 100%). As such, a method of theinvention provides the advantage that a substantially pure population ofa particular type of cells (e.g., SB-3 and SB-4 cells) can be obtainedwithout contamination by other cell types.

Various cell-containing samples from a subject can be used to preparethe somatic stem cells of this invention. In a preferred embodiment ofthis invention, SB-3 and SB-4 cells are prepared from the SB cellpopulation.

To confirm that the isolated cell is indeed SB-3 or SB-4 cell, one canexamine a number of characteristics, including cell surface markers.Antibodies against cell surface markers, such as CD10, CXCR4, CD31,CD105, CD44, and nestin, can be used. They can be conjugated withsuitable labels, such as fluorescein isothiocyanate (FITC),phycoerythrin (PE), or quantum dots.

To confirm the differentiation potential of these somatic stem cells,they can be induced to form, for example, neuro-glial cells, osteocytes,and adipocytes by methods known in the art. For example, these cells canbe passed and cultured to confluence, shifted to an osteogenic medium oran adipogenic medium, and incubated for a suitable period of time (e.g.,3 weeks). The differentiation potential for osteogenesis is assessed bythe mineralization of calcium accumulation, which can be visualized byvon Kossa staining. To examine adipogenic differentiation, intracellularlipid droplets can be stained by Oil Red O and observed under amicroscope. For neural differentiation, these cells can be incubated ina neurogenic medium for a suitable duration (e.g., 7 days), and thensubjected to serum depletion and incubation with β-mercaptoethanol.After differentiation, they exhibit the morphology of refractile cellbody with extended neurite-like structures arranged into a network. RTPCR and immunocytochemical stain of lineage specific markers are furtherconducted to confirm neural differentiation. Examples of the markersinclude neuron specific class III β-tubulin (Tuj-1), neurofilament, andGFAP.

SB-3 and SB-4 cells can be further propagated in a non-differentiatingmedium for more than 10, 20, 50, or 100 population doublings withoutindications of spontaneous differentiation, senescence, morphologicalchanges, increased growth rate, and changes in ability to differentiateinto neurons. These stem cells can be stored by standard methods beforeuse.

The terms “proliferation” and “expansion” as used interchangeably hereinwith reference to cells, refer to an increase in the number of cells ofthe same type by division. The term “differentiation” refers to adevelopmental process whereby cells become specialized for a particularfunction, for example, where cells acquire one or more morphologicalcharacteristics and/or functions different from that of the initial celltype. The term “differentiation” includes both lineage commitment andterminal differentiation processes. Differentiation may be assessed, forexample, by monitoring the presence or absence of lineage markers, usingimmunohistochemistry or other procedures known to a worker skilled inthe art. Differentiated progeny cells derived from progenitor cells maybe, but are not necessarily, related to the same germ layer or tissue asthe source tissue of the stem cells. For example, neural progenitorcells and muscle progenitor cells can differentiate into hematopoieticcell lineages.

The terms “lineage commitment” and “specification,” as usedinterchangeably herein, refer to the process a stem cell undergoes inwhich the stem cell gives rise to a progenitor cell committed to forminga particular limited range of differentiated cell types. Committedprogenitor cells are often capable of self-renewal or cell division.

The term “terminal differentiation” refers to the final differentiationof a cell into a mature, fully differentiated cell. For example, neuralprogenitor cells and muscle progenitor cells can differentiate intohematopoietic cell lineages, terminal differentiation of which leads tomature blood cells of a specific cell type. Usually, terminaldifferentiation is associated with withdrawal from the cell cycle andcessation of proliferation. The term “progenitor cell,” as used herein,refers to a cell that is committed to a particular cell lineage andwhich gives rise to cells of this lineage by a series of cell divisions.An example of a progenitor cell would be a myoblast, which is capable ofdifferentiation to only one type of cell, but is itself not fully matureor fully differentiated.

Within the scope of this invention is a cell bank or library having aplurality of populations of SB-3 or SB-4 cells. These stem cells can behuman cells or non-human cells. The bank can be produced by storingpopulations of SB-3 or SB-4 cells originated from different subjects;characterizing SB-3 or SB-4 cells in populations to obtain at least onepredetermined characteristic for each, and cataloguing each of thepopulations according to the at least one predetermined characteristic.To produce the bank, one can further expand the populations of SB-3 orSB-4 cells. Examples of the characteristics include a subject's name,gender, physical conditions (including genetic disorders and MHCinformation)

The above-described SB-3 cells and SB-4 cells can both be used in, amongothers, drug screening, treating degenerative disorders, and genetherapy.

Screening Methods

The above-described SB-3 cells and SB-4 cells can be used in screeningassays to identify drugs that can affect a particular cell type in amanner indicating that the drug can be useful for treating a disorderassociated with the cell type. For example, one can use the stem cellsin a method for identifying a drug candidate for treating a disease(e.g., a degenerative disease). The method includes the steps ofcontacting a test compound with the stem cells and determining theexpression level of a polypeptide that is down-regulated in the disease.The expression level in the presence of the test compound, if higherthan that in the absence of the compound, indicates that the compound isa candidate for treating the disease. Examples of the disease includediabetes, a neurodegenerative disease, arthritis, cancer, or anautoimmune disorder. The expression level can be determined at eitherthe mRNA level or at the protein level.

Thus, one aspect of the present invention relates to a method foridentifying an agent that alters a function of SB-3 or SB-4 cells bycontacting the cells with a test agent. A change in a function or geneexpression of the cells in presence of the test agent as compared tothat in the absence of the test agent indicates that the test agent isan agent that alters the function of or the gene expression in thecells. The term “test agent” refers to any molecule that is beingexamined for an ability to alter a function of or gene expression in thecells. Although the method generally is used as a screening assay toidentify previously unknown molecules that have a desired activity, thescreening methods of the invention also can be used to confirm aparticular activity of an agent known to have the activity.

The function can be expression of a gene that typically is expressed (ornot expressed) in the cells, and the agent can alter the function byincreasing or decreasing the level of expression of an expressed gene,or by turning on the expression of an unexpressed gene (e.g., inducingexpression of lineage-specific antigen) in the cells.

In one embodiment, the agent that affects a function of the cells is onethat induces differentiation of stem cells, thereby producingdifferentiated cells. Such differentiated cells can be multipotentialhuman stem cells (e.g., hematopoietic stem cells) or can be terminallydifferentiated cells (e.g., muscle cells, liver cells, neuronal cells,blood cells, connective tissue, or epithelial cells). As such, themethod can be used to identify an agent that induces differentiation ofSB-3 or SB-4 cells to terminally differentiated cells includingpancreatic beta cells, hepatocytes, cardiomyocytes, skeletal musclecells, or any other cell type. Agents or compound thus identified can beused to treat degenerative disorders, cancer, or immune disorders.

The expression level can be determined at either the mRNA level or theprotein level. Methods of measuring mRNA levels in a sample are wellknown in the art. To measure mRNA levels, cells can be lysed and thelevels of mRNA in the lysates, whether purified or not, can bedetermined by, e.g., hybridization assays (using detectably labeledgene-specific DNA or RNA probes) and quantitative or semi-quantitativeRT-PCR (using appropriate gene-specific primers). Alternatively,quantitative or semi-quantitative in situ hybridization assays can becarried out on tissue sections or unlysed cell suspensions usingdetectably (e.g., fluorescent or enzyme) labeled DNA or RNA probes.Additional mRNA-quantifying methods include the RNAse protection assay(RPA) method and the serial analysis of gene expression (SAGE) method,as well as array-based technologies.

Methods of measuring protein levels in a sample are also well known inthe art. Some of them employ antibodies (e.g., monoclonal or polyclonalantibodies) that bind specifically to a target protein. In such assays,the antibody itself or a secondary antibody that binds to it can bedetectably labeled. Alternatively, the antibody can be conjugated withbiotin. Its presence can be determined by detectably labeled avidin (apolypeptide that binds to biotin). Combinations of these approaches(including “multi-layer sandwich” assays) can be used to enhance thesensitivity of the methodologies. Appropriate labels includeradionuclides (e.g., ¹²⁵I, ¹³¹I, ³⁵S, ³H, or ³²P), enzymes (e.g.,alkaline phosphatase, horseradish peroxidase, luciferase, orβ-glactosidase), fluorescent/luminescent agents (e.g., fluorescein,rhodamine, phycoerythrin, GFP, BFP, and nanoparticles (e.g., Qdot™supplied by the Quantum Dot Corporation, Palo Alto, Calif.). Someprotein-measuring assays (e.g., ELISA and Western blot) can be appliedto body fluids or to cell lysates, and others (e.g., immunohistologicalmethods and fluorescence flow cytometry) can be applied to histologicalsections or unlysed cell suspensions. Other applicable methods includequantitative immunoprecipitation and complement fixation assays.

A test compound or agent can be any type of molecule, for example, apolynucleotide, a peptide, a peptidomimetic, peptoids such as vinylogouspeptoids, a small organic molecule, or the like, and can act in any ofvarious ways to alter a function of SB-3 or SB-4 cells. For example, thetest agent can act extracellularly by binding to a cell surface receptorexpressed by the cells, thereby altering a function mediated by bindingof a ligand that generally binds to and acts via the receptor.Alternatively, the test agent can be one that traverses the cellmembrane, either passively or via an active transport mechanism, andacts within the cells to alter a function. A peptide test agent can beany polymer of amino acids or amino acid analogs, and can vary fromabout three to four residues to hundreds or thousands. Peptide testagents can be prepared by chemical synthesis, or using methods ofprotein purification, followed by proteolysis and, if desired, furtherpurification by chromatographic or electrophoretic methods, or can beexpressed from an encoding polynucleotide. A peptide test agent can bebased on a known peptide, for example, a naturally occurring peptide,but can vary from the naturally occurring sequence, for example, bycontaining one or more amino acid analogs.

A polynucleotide agent can be a sequence of two or moredeoxyribonucleotides or ribonucleotides that are linked together by aphosphodiester bond. It can be RNA or DNA, which can be a gene or aportion thereof, a cDNA, an RNAi agent, a syntheticpolydeoxy-ribonucleic acid sequence, or the like, and can be singlestranded or double stranded, as well as a DNA/RNA hybrid. It can be anaturally occurring nucleic acid molecule, which can be isolated from acell, as well as a synthetic molecule, which can be prepared, forexample, by methods of chemical synthesis or by enzymatic methods suchas by the polymerase chain reaction (PCR). In various embodiments, apolynucleotide of the invention can contain nucleoside or nucleotideanalogs, or a backbone bond other than a phosphodiester bond. Suchnucleotide analogs are well known in the art and commercially available,as are polynucleotides containing such nucleotide analogs (Pagratis etal., Nature Biotechnol. 15:68-73, 1997).

A polynucleotide test agent can be contacted with or introduced intoSB-3 or SB-4 cells using methods as disclosed herein or otherwise knownin the art. Generally, but not necessarily, the polynucleotide isintroduced into the cell, where it effects its function either directly,or following transcription or translation or both. For example, thepolynucleotide can encode a peptide test agent, which is expressed inthe cells and alters a function of the cells. A polynucleotide testagent also can be, or can encode, an antisense molecule, a ribozyme or atriplexing agent, which can be designed to target one or more specifictarget nucleic acid molecules.

Candidate agents or compounds to be screened (e.g., proteins, peptides,peptidomimetics, peptoids, antibodies, small molecules, or other drugs)can be obtained using any of the numerous approaches in combinatoriallibrary methods known in the art. Such libraries include: peptidelibraries, peptoid libraries (libraries of molecules having thefunctionalities of peptides, but with a novel, non-peptide backbone thatis resistant to enzymatic degradation); spatially addressable parallelsolid phase or solution phase libraries; synthetic libraries obtained bydeconvolution or affinity chromatography selection; and the “one-beadone-compound” libraries. See, e.g., Lam, 1997, Anticancer Drug Des.12:145. Examples of methods for the synthesis of molecular libraries canbe found in, e.g., Angew. Chem. Int. Ed. Engl. 33:2061; and Gallop etal., 1994 J. Med. Chem. 37:1233. Libraries of compounds may be presentedin solution (e.g., Houghten, 1992, Biotechniques 13:412-421), or onbeads (Lam, 1991, Nature 354:82-84), chips (Fodor, 1993, Nature364:555-556), bacteria (U.S. Pat. No. 5,223,409), spores (U.S. Pat. No.5,223,409), plasmids (Cull et al., 1992, PNAS USA 89:1865-1869), orphages (Felici 1991, J. Mol. Biol. 222:301-310; and U.S. Pat. No.5,223,409).

Treatment Methods

One can use SB-3 or SB-4 cells disclosed herein for treatingdegenerative or inherited diseases, avoiding ethical considerations ofhuman embryo manipulation.

To do so, one can isolate a SB cell population from a patient, e.g., apatient lacking a functional gene essential for proper development of atissue or organ. The SB cell population is subsequently subjected toconditions so as to obtain SB-3 cells or SB-4 cells. One can thenintroduce into these stem cells an expression nucleic acid vectorencoding a functional version of the gene. The vector can be introducedinto the stem cells via a variety of techniques, including calciumphosphate co-precipitation, DEAE-dextran-mediated transfection,lipofection, electroporation, microinjection, or virus-meditatedtechniques. Methods not affecting the pluripotency of the cells arepreferred. Description of such techniques can be found in publications,e.g., U.S. Pat. Nos. 7,422,736 and 5,591,625. After delivering thefunctional gene into the stem cells, one can transplant them back intothe patient using method known in the art. As the stem cells areproduced from the patient, the treatment does not cause immunerejection.

Alternatively, one can make universal donor cells from SB-3 or SB-4cells prepared from a healthy subject. Methods for making universaldonor cells are known in the art. Exemplary techniques for makinguniversal pluripotent stem cells from SB-3 or SB-4 cells are describedbelow.

Under proper conditions, the transplanted stem cells can develop into afunctional tissue or organ. To facilitate this development, the patientmay be administered with factors to induce the development of the cells.Such factors can be small molecule compounds, peptides, and nucleicacids. Examples include, but are not limited to, transforming growthfactor β, bone morphogenic proteins, and nerve growth factor.

The universal pluripotent stem cells are also useful for studyingdevelopment or differentiation mechanisms of lineage development anddifferentiation. One can identify conditions for inducing thedevelopment of totipoent pluripotent stem cells into a specific tissueor organ using such cells as a model system. Further, one can isolategenes that play roles during the development using differential cDNAscreening known in the art. One can prepare a cDNA library from thecells that have been induced to develop into a certain lineage, e.g.,neuro-glial lineage described above. The library can then be used toisolate and study genes differentially expressed. These isolated genescan be further studied to define their roles in respective processes.The related techniques are known in the art. See e.g., U.S. Pat. No.7,422,736. The pluripotent stem cells can also be used to develop intoorgans or clones of the animals using the methods known in the art.Accordingly, these cells are valuable for the pet and livestockindustries, and can be used to preserve endangered animals.

In one aspect, the invention features a method of treating adegenerative disease in a subject. The method includes administering toa subject in need thereof an effective amount of the above-describedSB-3 or SB-4 cells. In one embodiment, at least one of these cellsincludes a recombinant nucleic acid. The recombinant nucleic acid canencode a polypeptide and the stem cell can contain an mRNA encoding thepolypeptide. Examples of the degenerative disease include a muscledegenerative disease, a liver degenerative disease, diabetes, aneurodegenerative disease, and arthritis. An example of aneurodegenerative disease is Parkinson's disease.

In another aspect, the invention features a method of treating anautoimmune disease in a subject. The method includes administering to asubject in need thereof an effective amount of the above-described SB-3or SB-4 cells.

A degenerative disease refers to a disorder where the function orstructure of an affected tissue or organ progressively deteriorate overtime, whether due to genetic defects, injury, lack of proper celldifferentiation (e.g., that in cell proliferative disorders), normalbodily wear, or lifestyle choices. Examples of degenerative diseasesinclude neurodegenerative diseases (e.g., Alzheimer's disease,Parkinson's disease, Huntington's disease, multiple sclerosis, andamyotrophic lateral sclerosis (ALS)); other nervous system disorders(e.g., transverse myelitis, demyelination occurring after trauma to thebrain or spinal cord, acute brain injury, head trauma, spinal cordinjury, peripheral nerve injury, ischaemic brain injury, hereditarymyelin disorder of the CNS, epilepsy, perinatal asphxia, asphyxia,anoxia, status epilepticus, Shy-Drager syndrome, autism, and stroke);cancer (e.g., liver cancer) or a condition resulting from anti-cancertherapy (e.g., chemotherapy); metabolic disorders (e.g.,diabetes/diabetes mellitus and Niemann Pick disease); autoimmune orinflammation related disorders (e.g., erythematosis, inflammatory boweldisease (IBD), prostatitis, osteoarthritis, osteoporosis, rheumatoidarthritis, lupus, diabetes, and asthma); ocular disorders (e.g.,glaucoma, retinitis pigmentosa, Norrie disease, and maculardegeneration); heart and circulatory disorders (e.g., atherosclerosis,heart failure myocardial infarction, and cardiovascular disease); blooddisorders (e.g., Wiscott Aldrich syndrome); muscular dystrophy;gastrointestinal disease; kidney disease; liver disease; lung disease;adrenal disorders (e.g., Addison's disease); a condition resulting froman injury (e.g., a burn, a stroke, damaged tissue including fleshwounds, age damaged cells, and age damaged tissue); a conditionassociated with aging (e.g., hair loss, including male pattern baldnessand alopecia areata); viral conditions (e.g., hepatitis C infection andacquired immune deficiency disorder); and any other disorder that anorgan transplant or stem cells can be used to restore, regenerate, orotherwise ameliorate signs and/or symptoms associated with the disorder.The method of this invention can be used in treating erectiledysfunction and in plastic surgery or breast implantation for female.

In yet another aspect, described herein a method of treating brain orCNS tissue damage or alleviating the symptom of the disorder in asubject. The method includes identifying a subject suffering from orbeing at risk for developing brain tissue damage. Examples of the braintissue damage include those caused by a cerebral ischemia (e.g., chronicstroke) or a neurodegenerative disease (e.g., Parkinson's disease,Alzheimer's disease, Spinocerebellar disease, and Huntington's disease).The treatment method entails administering to a subject in need thereofan effective amount of the above-described stem cells or activeagents/compounds.

The therapeutic effects of the stem cells can be accessed according tostandard methods. For example, to confirm efficacy in promotingcerebrovascular angiogenesis, one can examine the subject before andafter the treatment by standard brain imaging techniques, such ascomputed tomography (CT), Doppler ultrasound imaging (DUI), magneticresonance imaging (MRI), and proton magnetic resonance spectroscopy(¹H-MRS). For example, ¹H-MRS represents a non-invasive means to obtainbiochemical information correlated to brain metabolic activity (Lu etal., 1997, Magn. Reson. Med. 37, 18-23). This technique can be appliedto evaluate the metabolic changes involved in cerebral ischemia with orwithout stem cell transplantation. For example, it can be used to studythe N-acetylaspartate (NAA) concentration in the brain, a marker ofneuronal integrity. Although NAA redistribution and trapping in neuronaldebris limits its use as a quantitative neuronal marker, decreases inbrain NAA concentration in cerebral ischemia can be considered as anindex of neuronal loss or dysfunction (Demougeot et al., 2004, J.Neurochem. 90, 776-83). Therefore, an NAA level, measured by ¹H-MRS, isa useful indicator for following the effect of stem cell transplantationafter cerebral ischemia.

A subject to be treated for one of the above-described disorders can beidentified by standard diagnosing techniques for that particulardisorder. “Treating” refers to administration of a composition (e.g., acell composition) to a subject, who is suffering from or is at risk fordeveloping that disorder, with the purpose to cure, alleviate, relieve,remedy, delay the onset of, prevent, or ameliorate the disorder, thesymptom of the disorder, the disease state secondary to the disorder, orthe predisposition toward the damage/disorder. A subject to be treatedcan be identified by standard techniques for diagnosing the conditionsor disorders of interest. An “effective amount” refers to an amount ofthe composition that is capable of producing a medically desirableresult in a treated subject. The treatment method can be performed aloneor in conjunction with other drugs or therapies. The subject can be ahuman or a non-human mammal, such as a cat, a dog, or a horse.

Making and Using Liver Cells

Also within the scope of this invention are methods of making and usingliver cells derived from SB-4 cells. SB-4 cells undergo differentiationto form endoderm cells when cultured in a medium containing activin forfive days, then in a medium containing bFGF and BMP2 for ten to fifteendays, finally in a medium containing HGF, DEX, and OSM for ten tofifteen days. These endoderm cells express albumin, transferrin, andHNF3B, all of which are liver-specific proteins. In other words, SB-4cells can differentiate into liver cells for use in producing albumin orfor use in a bioartificial liver device. Herein, the term “liver cells”is interchangeable with the word “hepatocytes.”

One embodiment of this invention is an extracorporeal bioartificialliver device that uses the liver cells derived from SB-4 cells. Such adevice is used to treat a subject having or suspected of having aliver-related condition or compromised liver function resulting eitherfrom disease or trauma.

The extracorporeal bioartificial liver device includes one or morecartridges. See FIG. 2. Each cartridge contains an array of hollowfibers each formed of a permeable or semi-permeable membrane. See FIGS.3a and 3b . Each cartridge can have a cylindrical shape with a firstopening at one terminus and a second opening at the other terminus; andthe first opening is affixed to a first passage and the second openingis affixed to a second passage, the two passages extending away from thecartridge. See FIG. 3a . One passage is attached to an artery of asubject and the other to a vein of this subject.

The membrane forming each hollow fiber allows cleansing of blood bypermitting crossover of toxic solutes from the blood to the liver cellscultured inside the hollow fibers (e.g., bilirubin, which diffusesthrough the membrane and are taken up and metabolized) and also bypermitting diffusion of vital metabolites from cells cultured in thedevice to the blood returning to the subject. The selectively permeableor semi-permeable character of the membrane also provides a mechanicalbarrier to components of the immune system in the blood of the subject.Typically, the membrane features a molecular weight cutoff from about20,000 daltons up to about 80,000 daltons, generally about 30,000daltons to about 50,000 daltons. In a preferred embodiment, the membranehas pores of 0.1 μm to 0.3 μm in diameter, typically about 0.2 μm. Apore diameter in this range, while excluding cellular elements, permitsproteins (e.g., albumin) and protein complexes to pass through, thusameliorating serum protein deficiencies of a subject suffering fromliver failure.

The term “cleanse” or “clean,” as used herein, means removal of unwantedor undesired molecules from the subject's blood. Moreover, the term“cleanse” or “clean” further includes release of desired molecules(i.e., albumin) from the SB-4-derived liver cells into the blood beforereturning it to the subject.

Similar devices, their use, and mechanisms of action are commonly knownto a person of ordinary skill in the art. For example, bio-artificialliver devices are described in Viles, et al., U.S. Pat. Nos. 4,675,002and 4,853,324; Jauregin, GB 2,221,857A; Wolf et al., 1979, InternationalJ. of Artificial Organs 2:97-103; Wolf et al., 1978, International J. ofArtificial Organs, 1:45-51; and Ehrlich et al., 1978, In Vitro14:443-450. Such devices are also contemplated to be used with the SB-4cells-derived liver cells.

Hollow fiber cartridges have two-chamber units, i.e., one chamberlocated inside each of the hollow fibers and the other located outsidethe hollow fibers, which reproduce the three-dimensional characteristicsof normal organs. See Knazek, R. H., 1974, Feder. Proc. 33:1978-1981;and Ku, K. et al., 1983, Biotechnol. Bioeng. 23:79-95.

Culture or growth medium is circulated through the capillary spaceinside each of the hollow fibers in the cartridge and the cells, grownin the extracapillary space between the hollow fibers in the cartridgeafter seeding, are supplied with a constant inflow of fresh medium. SeeTharakan, J. P. et al., 1986, Biotechnol. Bioeng. 28:1605-1611.Typically, 1400 cm² cartridges are inoculated with an effective numberof SB-4 cell-derived liver cells (e.g., approximately, 1×10⁹ cells) andgrown to confluence in 14 to about 21 days. Such hollow fiber culturesystems are well known (e.g., Heifetz et al., 1989, BioTechniques7:192-199; and Donofrio, D., Amer. Biotech. Lab. Sept. 1989, Publication#940) and available commercially (e.g., the Anchornet series).

Hollow fiber-based systems when used in a bio-artificial liver deviceoffer several advantages. Cartridges support the growth of veryhigh-density cultures. Based on the extracapillary volume, 15 to 20 g ofcells are grown in a 1400 cm² unit and 100 g of cells are grown in a7000 cm². This amount of cell mass is capable of providing liver supportto a subject suffering from liver failure. Also, cartridge-grown cellsare polarized and their growth approximates normal liver structure. Thecells receive nutrients from the capillary space and secrete wasteproducts into the extracapillary space. The extracapillary space isperfused to prevent the accumulation of toxic products. The continualflow of media and the in-line oxygenator provide a more constant supplyof oxygen and energy.

For the most part, the bio-artificial liver devices contemplated to beused with the SB-4-derived liver cells primarily process blood byextracorporeally attaching to a subject (e.g., typically, making fluidcommunication from the device to the subject's blood supply usuallybetween an artery and a vein). Such an arrangement is particularlyuseful for providing temporary liver support for subjects suffering froma severe liver disorder.

Alternatively, the SB-4-derived liver cells are used within the body asa bio-artificial liver or as a bio-artificial liver support. When usedin this manner, the SB-4-derived liver cells are encapsulated or grownin hollow fiber capillary membranes for internal use. Typically, thecells attach to the support upon growth. Linkage materials, however, maybe provided to attach the cells to a support (see, e.g., Jauregin, GB2,221,857A). The SB-4-derived liver cells are encapsulated inbiomaterial such as alginate-polylysine membranes, as taught by Cal etal., Artificial Organs 12:388-393; Sun et al., 1986, Trans. Am. Soc.Artif. Intern. Organs Vol. XXXII:39-41; O'Shea et al., 1984, BiochimicaBiophysica Acta 804:133-136; Sun et al., 1985, J. Controlled Release2:137-141; and Lim, U.S. Pat. No. 4,391,909. The encapsulated cells andvehicle capsules are then injected intra-peritoneally into the subject.

Additionally, the SB-4-derived liver cells can be used in a syntheticliver-like tissue comprising fibroblasts and the SB-4-derived livercells. Typically, co-culture of fibroblasts and hepatocytes do notautomatically adopt the arrangement typically found in the liver.Further, as the two cell types communicate poorly, the hepatocytes areoften functionally inefficient. To solve this problem, one can imprint asubstrate (e.g., a borosilicate wafer) using standard photolithographictechniques of microelectronic technology with patterned films ofcollagen, which promotes cell adhesion. See Toner et al., 1997, FallMeeting of the Materials Research Association, 1-5; Toner et al., 1988,Nature, 39: 128. The SB-4-derived liver cells can then be cultured onsuch surfaces, adhering only to the collagen-coated regions. Fibroblastsare subsequently introduced onto the bare surface regions producing anintimate mixture of the two cell types in a periodic pattern. Thistechnique allows any ratio of cell types on the substrate and thuspermits adjustment to any physiological value. Further, any patternsize, shape, and number density can be deduced and engineered.

Gene Therapy

The stem cells described herein can be used to express an exogenous,recombinant polypeptide. Thus, within the scope of this invention aresuch stem cells, which contain a recombinant nucleic acid. Therecombinant nucleic acid can encode a polypeptide and the stem cells cancontain an mRNA encoding the polypeptide.

These stem cells can be genetically manipulated so that they do notexpress the beta2-microglobulin gene or do not express one or moreproteins encoded by the class I major histocompatibility complex (MHC)genes that elicit a T lymphocyte mediated reaction against the cell.These cells can be used as universal donor cells since they do not leadto host rejections of grafts.

Accordingly, the invention features a method for introducing aheterologous nucleic acid in a subject. The method includes the steps ofobtaining the above-described stem cells, where at least one of the stemcells includes a heterologous nucleic acid, and administering the cellinto a subject in need thereof. The heterologous nucleic acid can encodea polypeptide.

The term “heterologous” is a relative term, which when used withreference to portions of a nucleic acid indicates that the nucleic acidcomprises two or more subsequences that are not found in the samerelationship to each other in nature. For instance, a nucleic acid thatis recombinantly produced typically has two or more sequences fromunrelated genes synthetically arranged to make a new functional nucleicacid, e.g., a promoter from one source and a coding region from anothersource. The two nucleic acids are thus heterologous to each other inthis context. When added to a cell, the recombinant nucleic acids wouldalso be heterologous to the endogenous genes of the cell. Thus, in achromosome, a heterologous nucleic acid would include a non-native(non-naturally occurring) nucleic acid that has integrated into thechromosome, or a non-native (non-naturally occurring) extrachromosomalnucleic acid. In contrast, a naturally translocated piece of chromosomewould not be considered heterologous in the context of this patentapplication, as it comprises an endogenous nucleic acid sequence that isnative to the mutated cell. Similarly, a heterologous protein indicatesthat the protein comprises two or more subsequences that are not foundin the same relationship to each other in nature (e.g., a “fusionprotein,” where the two subsequences are encoded by a single nucleicacid sequence). Such protein can be generated by recombinant techniques.

The term “recombinant” when used with reference, e.g., to a cell,nucleic acid, protein, or vector, indicates that the cell, nucleic acid,protein, or vector, has been modified by the introduction of aheterologous nucleic acid or protein or the alteration of a nativenucleic acid or protein, or that the cell is derived from a cell somodified. Thus, for example, recombinant cells express genes that arenot found within the native (naturally occurring) form of the cell orexpress a second copy of a native gene that is otherwise normally orabnormally expressed, under expressed or not expressed at all.

The above-described stem cells and methods can be used in various genetherapy methods known in the art. Gene therapy includes both ex vivo andin vivo techniques. Specifically, the above-described stem cells can begenetically engineered ex vivo with an oligonucleotide modulator or anucleic acid molecule encoding the modulator, with the engineered cellsthen being provided to a patient to be treated. Cell cultures may beformulated for administration to a patient, for example, by dissociatingthe cells (e.g., by mechanical dissociation) and intimately admixing thecell with a pharmaceutically acceptable carrier (e.g., phosphatebuffered saline solution). Alternatively, cells may be cultured on asuitable biocompatible support and transplanted into a patient. Theengineered cells are typically autologous so as to circumvent xenogeneicor allotypic rejection. Such ex vivo methods are well known in the art.

The cells can be engineered by administration of the oligonucleotide ornucleic acid molecule using techniques known in the art. For example,oligonucleotides and other nucleic acid molecules can be administered bydirect injection of a “naked” nucleic acid molecule (U.S. Pat. No.5,679,647) or a nucleic acid molecule formulated in a composition withone or more other agents which facilitate uptake of the nucleic acidmolecule by the cell, such as saponins (see, for example, U.S. Pat. No.5,739,118) or cationic polyamines (see, for example, U.S. Pat. No.5,837,533); by microparticle bombardment (for example, through use of a“gene gun”; Biolistic, Dupont); by coating the nucleic acid moleculewith lipids, cell-surface receptors or transfecting agents; byencapsulation of the nucleic acid molecule in liposomes, microparticles,or microcapsules; by administration of the nucleic acid molecule linkedto a peptide which is known to enter the nucleus; or by administrationof the nucleic acid molecule linked to a ligand subject toreceptor-mediated endocytosis, which can be used to target cell typesspecifically expressing the receptors.

A nucleic acid-ligand complex can be formed in which the ligandcomprises a fusogenic viral peptide to disrupt endosomes, allowing thenucleic acid to avoid lysosomal degradation; or the nucleic acidmolecule can be targeted for cell specific uptake and expression in vivoby targeting a specific receptor. In addition, an efficient method forthe introduction, expression and accumulation of antisenseoligonucleotides in the cell nucleus is described in U.S. Pat. No.6,265,167, which allows the antisense oligonucleotide to hybridise tothe sense mRNA in the nucleus, and thereby prevents the antisenseoligonucleotide being either processed or transported into thecytoplasm. The present invention also contemplates the intracellularintroduction of the nucleic acid molecule and subsequent incorporationwithin host cell DNA for expression by homologous recombination known inthe art.

The polynucleotide can also be incorporated into a suitable expressionvector. A number of vectors suitable for gene therapy applications areknown in the art (see, for example, Viral Vectors: Basic Science andGene Therapy, Eaton Publishing Co. (2000)).

The expression vector may be a plasmid vector. Methods of generating andpurifying plasmid DNA are rapid and straightforward. In addition,plasmid DNA typically does not integrate into the genome of the hostcell, but is maintained in an episomal location as a discrete entityeliminating genotoxicity issues that chromosomal integration may raise.A variety of plasmids are now readily available commercially and includethose derived from Escherichia coli and Bacillus subtilis, with manybeing designed particularly for use in mammalian systems. Examples ofplasmids that may be used in the present invention include, but are notlimited to, the eukaryotic expression vectors pRc/CMV (Invitrogen),pCR2.1 (Invitrogen), pAd/CMV and pAd/TR5/GFPq (Massie et al., (1998)Cytotechnology 28:53-64). In an exemplary embodiment, the plasmid ispRc/CMV, pRc/CMV2 (Invitrogen), pAdCMV5 (MB-NRC), pcDNA3 (Invitrogen),pAdMLP5 (IRB-NRC), or PVAX Invitrogen).

The expression vector can be a viral-based vector. Examples ofviral-based vectors include, but are not limited to, those derived fromreplication deficient retrovirus, lentivirus, adenovirus andadeno-associated virus. Retrovirus vectors and adeno-associated virusvectors are currently the recombinant gene delivery system of choice forthe transfer of exogenous oligonucleotides or genes in vivo,particularly into humans. These vectors provide efficient delivery ofgenes into cells, and the transferred nucleic acids are stablyintegrated into the chromosomal DNA of the host. A major prerequisitefor the use of retroviruses is to ensure the safety of their use,particularly with regard to the possibility of the spread of wild-typevirus in the cell population. Retroviruses, from which retroviralvectors may be derived include, but are not limited to, Moloney MurineLeukemia Virus, spleen necrosis virus, Rous Sarcoma Virus, HarveySarcoma Virus, avian leukosis virus, gibbon ape leukemia virus, humanimmunodeficiency virus, adenovirus, Myeloproliferative Sarcoma Virus,and mammary tumor virus. Specific retroviruses include pLJ, pZIP, pWEand pEM, which are well known to those skilled in the art.

Cell Banking

The invention features a stem cell bank or library for a convenientsystematic access to different stem cell lines. SB-3 or SB-4 cells inthe bank or library are derived from a healthy subject or subject havingknown disease state or disease symptom would be invaluable to users,e.g., researchers. Also with the scope of the invention is a cell bankor library having cells differentiated from the above-described stemcells. Examples of cells differentiated from the stem cells includebrain cells, neurons, astrocytes, glial cells, T cells, B cells,cartilage cells, bone cells, pancreatic islet cells, fat cells, heartcells, liver cells, kidney cells, lung cells, muscle cells, and eyecells. The subjects may be human or nonhuman vertebrates. The stem cellscan be derived from any mammalian organism, such as human, mouse,rabbits, cows, pigs, and the like.

The cells in the bank or library are catalogued according topredetermined characteristics, including phenotypic information,morphological characteristics, differentiation profile, blood type,major histocompatibility complex, disease state of donor, or genotypicinformation (e.g. single nucleated polymorphisms (SNPs) of a specificnucleic acid sequence associated with a gene, or genomic ormitochondrial DNA). The cells are stored under appropriate conditions(typically by freezing) to keep the stem cells alive and functioning.Cataloguing may constitute creating a centralized record of thecharacteristics obtained for each cell population, such as, but notlimited to, an assembled written record or a computer database withinformation inputted therein. Essentially, this embodiment pertains tothe production of a stem cell bank. The stem cell bank facilitates theselection from a plurality of samples of a specific stem cell samplesuitable for a user's needs. Thus, another embodiment of the subjectinvention pertains to a stem cell bank comprising a plurality of stemcells samples obtained from separate sources and which are characterizedand catalogued according to at least one predetermined characteristic.An additional embodiment pertains to a method of establishing a stemcell bank comprising collecting stem samples from multiple sources;cataloguing the samples according to at least one predeterminedcharacteristic and storing the cells under conditions that keep cellsviable.

With the scope of this invention is a stem cell banking systemcontaining a plurality of stem cell populations disposed in individualcontainers under conditions to keep the stem cell populations viable; adatabase computer comprising at least one processing module, a display,and a storage medium comprising information of at least onecharacteristic for each stem cell population; and at least one programcode module for causing the information to be viewable on said displayupon command by a user. In a specific embodiment, the invention featuresa stem cell banking system where stem cell populations have stem cellsobtained from subjects who have a disease condition. The diseasecondition may include the above-described degenerative diseases. SB-3 orSB-4 cells derived from different subjects having different diseases,and the stem cells are characterized. The characteristic(s) is/areinputed into the database computer. In addition, or alternatively, cellsare characterized based on a specific phenotype not necessarilyassociated with a disease condition. For example, liver cells can becharacterized based on their ability to metabolize certain compoundssuch as caffeine, alcohol, drug agents, etc. to study genetic bases ofsuch different metabolism abilities, or underlying physiology associatedtherewith. Other types of cells can be characterized based on functionaland/or morphological phenotypes.

In certain embodiments, cells differentiated from SB-3 or SB-4 cells maybe subjected to conditions to influence differentiation ordedifferentiation through introduction of engineered vectors, or othergenetic material. Dedifferentiation comprises the manipulation of a cellsuch that it takes on the properties of a less differentiated cell.

The stem cell libraries of the invention can be used to screen foragents or compounds that may be used to treat degenerative disorders,cancer or immune disorders in the manner described above. The librariesare suitable for high throughput screening and are useful foridentifying agents that are specifically effective for a particularsubject. For a high throughput screening, stem cells can be introducedinto wells of a multiwell plate or of a glass slide or microchip, andcan be contacted with the test agent. Generally, the cells are organizedin an array, particularly an addressable array, such that roboticsconveniently can be used for manipulating the cells and solutions andfor monitoring the cells, particularly with respect to the functionbeing examined. An advantage of using a high throughput format is that anumber of test agents can be examined in parallel, and, if desired,control reactions also can be run under identical conditions as the testconditions. As such, the screening methods of the invention provide ameans to screen one, a few, or a large number of test agents in order toidentify an agent that can alter a function of stem cells, for example,an agent that induces the cells to differentiate into a desired celltype, or that prevents spontaneous differentiation, for example, bymaintaining a high level of expression of regulatory molecules.

Universal Donor Cells

The above-described stem cells can be genetically engineered to generatehistocompatible donor cells or tissues for transplantation. The goal oftransplantation and cell therapy is to successfully replace failingtissues or organs with functional donor tissues or organs. However, fortransplantation to succeed, two major barriers need to be overcome: theavailability of suitable donor tissues or organs and immune rejection.The replacement of failing tissues or organs and the treatment of therejection is restricted by the limited number of acceptable donors andthe need for co-administration of toxic immuno-suppressive drugs inconjunction with long term immuno-suppressive protocols. Current andexperimental transplantation protocols rely mainly on sibling donors,other small pools of allogeneic donors, and xenogeneic donors. Theabove-described genetically engineered stem cells can be used toovercome these limitations.

More specifically, the stem cells described herein can be geneticallyengineered to not express on their surface class II MHC molecules. Morepreferably, the cells are engineered to not express substantially allcell surface class I and class II MHC molecules. As used herein, theterm “not express” means either that an insufficient amount is expressedon the surface of the cell to elicit a response or that the protein thatis expressed is deficient and therefore does not elicit a response.

The MHC molecules refer to HLA molecules, specifically of classes HLA A,B and C, and class II HLA DP, DQ, and DR, and their subclasses. Thisterminology is generally construed as specific to the human MHC, but isintended herein to include the equivalent MHC genes from the donor cellspecies, for example, if the cells are of porcine origin, the term HLAwould refer to the equivalent porcine MHC molecules, whether MHC I orII. When the class II MHC molecules are removed, CD4+ T-cells do notrecognize the genetically engineered endothelial cells; when both theclass I and class II MHC molecules are removed neither CD4+ nor CD8+cells recognize the modified cells.

The preferred genetic modification performed on the stem cellsincludes 1) disrupting the endogenous invariant chain gene whichfunctions in the assembly and transport of class II MHC molecules to thecell surface and loading of antigenic peptide, and 2) disrupting theendogenous β₂-microglobulin gene (β₂M gene), which codes for a proteinrequired for the cell surface expression of all class I MHC molecules.Alternatively, just the invariant chain gene is disrupted. Invariantchain is believed to be required for the insertion of antigenic peptidefragments into the MHC class II molecule. Together, the antigenicpeptide and MHC are recognized by T cells. In the absence of antigenicpeptide, T cell recognition is not normally obtained, nor is the MHCclass II molecule folded properly. Thus, in cells lacking invariantchain, presentation of peptide will be abrogated and even if minusculeamounts of cell surface MHC are obtained, they may be devoid of peptideand therefore, non-immunogenic.

Disruption of these genes can be accomplished by means of homologousrecombination gene targeting techniques. These techniques are well knownin the art. See, e.g., U.S. Pat. Nos. 6,916,654 and 6,986,887.

Compositions

The present invention provides for pharmaceutical compositionscontaining the SB-3 or SB-4 cells or active agents/compounds.Pharmaceutical compositions can be prepared by mixing a therapeuticallyeffective amount of the cells or active agents/compounds, and,optionally other active substance, with a pharmaceutically acceptablecarrier. The carrier can have different forms, depending on the route ofadministration. Examples of other active substance include activecompounds known or identified by the screening method of describedabove.

The above-described pharmaceutical compositions can be prepared by usingconventional pharmaceutical excipients and methods of preparation. Allexcipients may be mixed with disintegrating agents, solvents,granulating agents, moisturizers, and binders. As used herein, the term“effective amount” or ‘therapeutically effective amount’ refers to anamount which results in measurable amelioration of at least one symptomor parameter of a specific disorder. A therapeutically effective amountof the above-described stem cells can be determined by methods known inthe art. An effective amount for treating a disorder can easily bedetermined by empirical methods known to those of ordinary skill in theart. The exact amount to be administered to a patient will varydepending on the state and severity of the disorder and the physicalcondition of the patient. A measurable amelioration of any symptom orparameter can be determined by a person skilled in the art or reportedby the patient to the physician. It will be understood that anyclinically or statistically significant attenuation or amelioration ofany symptom or parameter of the above-described disorders is within thescope of the invention. Clinically significant attenuation oramelioration means perceptible to the patient and/or to the physician.

The phrase “pharmaceutically acceptable” refers to molecular entitiesand other ingredients of such compositions that are physiologicallytolerable and do not typically produce unwanted reactions whenadministered to a human. Preferably, the term “pharmaceuticallyacceptable” means approved by a regulatory agency of the federal or astate government or listed in the U.S. Pharmacopeia or other generallyrecognized pharmacopeia for use in mammals, and more particularly inhumans. Pharmaceutically acceptable salts, esters, amides, and prodrugsrefers to those salts (e.g., carboxylate salts, amino acid additionsalts), esters, amides, and prodrugs which are, within the scope ofsound medical judgment, suitable for use in contact with the tissues ofpatients without undue toxicity, irritation, allergic response, and thelike, commensurate with a reasonable benefit/risk ratio, and effectivefor their intended use.

A carrier applied to the pharmaceutical compositions described aboverefers to a diluent, excipient, or vehicle with which a compound isadministered. Such pharmaceutical carriers can be sterile liquids, suchas water and oils. Water or aqueous solution, saline solutions, andaqueous dextrose and glycerol solutions are preferably employed ascarriers, particularly for injectable solutions. Suitable pharmaceuticalcarriers are described in “Remington's Pharmaceutical Sciences” by E. W.Martin, 18th edition.

The above-described stem cells can be administered to individualsthrough infusion or injection (for example, intravenous, intrathecal,intramuscular, intraluminal, intratracheal, intraperitoneal, orsubcutaneous), orally, transdermally, or other methods known in the art.Administration may be once every two weeks, once a week, or more often,but frequency may be decreased during a maintenance phase of the diseaseor disorder.

Both heterologous and autologous cells can be used. In the former case,HLA-matching should be conducted to avoid or minimize host reactions. Inthe latter case, autologous cells are enriched and purified from asubject and stored for later use. The cells may be cultured in thepresence of host or graft T cells ex vivo and re-introduced into thehost. This may have the advantage of the host recognizing the cells asself and better providing reduction in T cell activity.

The dose and the administration frequency will depend on the clinicalsigns, which confirm maintenance of the remission phase, with thereduction or absence of at least one or more preferably more than oneclinical signs of the acute phase known to the person skilled in theart. More generally, dose and frequency will depend in part on recessionof pathological signs and clinical and subclinical symptoms of a diseasecondition or disorder contemplated for treatment with theabove-described composition. Dosages and administration regimen can beadjusted depending on the age, sex, physical condition of administeredas well as the benefit of the conjugate and side effects in the patientor mammalian subject to be treated and the judgment of the physician, asis appreciated by those skilled in the art. In all of theabove-described methods, the stem cells can be administered to a subjectat 1×10⁴ to 1×10¹⁰/time.

The specific examples below are to be construed as merely illustrative,and not limitative of the remainder of the disclosure in any waywhatsoever. Without further elaboration, it is believed that one skilledin the art can, based on the description herein, utilize the presentinvention to its fullest extent. All publications cited herein arehereby incorporated by reference in their entirety. Further, anymechanism proposed below does not in any way restrict the scope of theclaimed invention.

EXAMPLE 1

A blood sample or a bone marrow sample was drawn from a person andplaced in an anti-clotting EDTA tube or heparin tube. After allowing thetube to rest for 72 hours at 4° C., the sample separated into twolayers. The bottom layer, which appeared red, consisted almost entirelyof red (RBC) and white blood cells. The top layer contained cells havingdiameters of less than 6 μm, named SB cells. SB cells are described infurther detail in US2012/0034194.

SB cells were then cultured in a StemPro-34 medium (INVITROGEN)containing 5 ng/ml R-Spondin-1, 5 ng/ml SCF, 5 ng/ml G-CSF, 20 ng/mlbFGF, 20 ng/ml EGF, and 5 ng/ml PDGF for 4 to 14 days. Under theabove-mentioned culturing condition, the diameters of the cellsincreased to 6-25 μm. The cells that remained non-adherent were namedSB-3 cells, and those that became adherent were named SB-4 cells. SB-4cells are round or oval, and larger in size, i.e., 7-30 μm, than SB-3cells.

CD349 antibody was used to isolate CD349+ cells from the SB mixture.Also see US2012/0034194. These CD349+cells cultured in the same way asSB cells also grew into SB-3 cells and attach to the walls as SB-4cells.

Tests showed that SB-3 cells have the ability to expand. SB-4 cells wereable to undergo proliferation and differentiation as long as the desiredgrowth factor was added. SB-4 cells were able to undergo at least 40passages with normal karyotype and normal telomerase activity. CulturedSB-4 cells had a doubling time around 24 hours.

RT-PCR analyses show that SB-3 cells expressed CD10, CXCR4, and CD31,but not CD9, CD349, CD271, CD133, CD66e, CD45, CD20, or CD4. SB-4 cellsexpressed CD105, CD44, and nestin, but not CD34, CD90, CD41, CD117,α-fetoprotein, POU5F1, Nanog, Sox2, HNF4a, CD36, HNF3B, Pax6, or Pax7.The primers for RT-PCR are shown in Table 1 below:

TABLE 1 a-feto- F: 5′- AAATGCGTTTCTCGTTGCTT-3′ protein (SEQ ID NO: 1)R: 5′- GCCACAGGCCAATAGTTTGT-3′ (SEQ ID NO: 2) B-actinF: 5′- AGCCTCGCCTTTGCCGA-3′ (SEQ ID NO: 3) R: 5′- CTGGTGCCTGGGGCG-3′(SEQ ID NO: 4) CD10 F: 5′- GGT TGG GAG CTG ATG AAA CT-3′ (SEQ ID NO: 5)R: 5′- GAA TAG GGC TGG AAC AAG GA-3′ (SEQ ID NO: 6) CD105F: 5′- CAC TAG CCA GGT CTC GAA GG-3′ (SEQ ID NO: 7)R: 5′- CTG AGG ACC AGA AGC ACC TC -3′ (SEQ lD NO: 8) CD117F: 5′- TAAGTCAGATGCGGCCATGACTGT 3′ (SEQ ID NO: 9)R: 5′- TGGTGCAGGCTCCAAGTAGATTCA -3′ (SEQ ID NO: 10) CD133F: 5′- AGC GAT CAA GGA GAC CAA AG-3′ (SEQ ID NO: 11)R: 5′- AAG CAC AGA GGG TCA TTG AG-3′ (SEQ ID NO: 12) CD271F: 5′- CCG CAA AGC GGA CCG AGC TG-3′ (SEQ ID NO: 13)R: 5′- CGT CAC GCT GTC CAG GCA GG-3′ (SEQ ID NO: 14) CD31F: 5′-CACAACAGACATGGCAACAAGGCT- 3′ (SEQ ID NO: 15)R: 5′-TCCTTCTGGATGGTGAAGTTGGCT-3′ (SEQ ID NO: 16) CD34F: 5′- CCT TGA ACC ACT TCC CTC AT-3′ (SEQ ID NO: 17)R: 5′- TAG GCT CCA GCC AGA AAA CT-3′ (SEQ ID NO: 18) CD349F: 5′- TCT TCC ACA TCC GCA AGA TCA-3′ (SEQ ID NO: 19)R: 5′- AGT CCA TGT TGA GGC GTT CGT A-3′ (SEQ ID NO: 20) CD36F: 5-CAGGAGTTTGCAAGAAACAGGTGC- 3′ (SEQ ID NO: 21)R: 5′- ATACCTCCAAACACAGCCAGGACA -3′ (SEQ ID NO: 22) CD4F: 5′-GTA GTA GCC CCT CAG TGC AA-3′ (SEQ ID NO: 23)R: 5′- AAA GCT AGC ACC ACG ATG TC-3′ (SEQ ID NO: 24) CD41F: 5′- AAT GGC CCC TGC TGT CGT GC-3′ (SEQ ID NO: 25)R: 5′- TGC ACG GCC AGC TCT GCT TC-3′ (SEQ ID NO: 26) CD44F: 5′-TCGAAGAAGGTGTGGGCAGAAGA -3′ (SEQ ID NO: 27)R: 5′-ATTTCCTGAGACTTGCTGGCCTCT -3′ (SEQ ID NO: 28) CD66eF: 5′- TAT ACG TGC CAA GCC CAT AA-3′ (SEQ ID NO: 29)R: 5′- TAC AGC ATC CTC ATC CTC CA-3′ (SEQ ID NO: 30) CD9F: 5′- TGC GTT GAA CTG CTG TGG TTT G-3′ (SEQ ID NO: 31)R: 5′- GCG CCG ATG ATG TGG AAT TT-3′ (SEQ ID NO: 32) CD90F: 5′- CATAACGCTCTCACCCTCTC-3′ (SEQ ID NO: 33)R: 5′- CTCTTCACCCCATTCACACC-3′ (SEQ ID NO: 34) CXCR4F: 5′- GTT GGC TGA AAA GGT GGT CT-3′ (SEQ ID NO: 35)R: 5′- CAC AAC CAC CCA CAA GTC AT-3′ (SEQ ID NO: 36) GAPDHF: 5′- GAG TCA ACG GAT TTG GTC GT-3′ (SEQ ID NO: 37)R: 5′- TTG ATT TTG GAG GGA TCT CG-3′ (SEQ ID NO: 38) HNF3BF: 5′- CCATGCACTCGGCTTCCAGTATG-3′ (SEQ ID NO: 39)R: 5′- CGCCGACATGCTCATGTACGTG-3′ (SEQ ID NO: 40) HNF4aF:5′- TGTGAGTGGCCCCGACCCTG-3′ (SEQ ID NO: 41)R: 5′- ACGATTGTGGCGACGGCTCC-3′ (SEQ ID NO: 42) CD20 set 1F: 5′- GCT GCC ATT TCT GGA ATG AT-3′ (SEQ ID NO: 43)R: 5′- TTC CTG GAA GAA GGC AAA GA-3′ (SEQ ID NO: 44) CD20 set 2F: 5′- GTT TTT GGT GGA GTC CCT TT-3′ (SEQ ID NO: 45)R: 5′- AAA CAG ATG GGT GTT GGC TA-3′ (SEQ ID NO: 46) NanogF: 5′- TGT GAT TTG TGG GCC TGA AGA-3′ (SEQ ID NO: 47)R: 5′- TTG TTT GCC TTT GGG ACT GG-3′ (SEQ ID NO: 48) NestinF: 5′- TGCCCGGCACTGGGGACTTA-3′ (SEQ ID NO: 49)R: 5′- TAGCGGGCCAGGCCTCTCAG-3′ (SEQ ID NO: 50) Pax 7F: 5′- CGA CTC CGG ATG TAG AGA AA-3′ (SEQ ID NO: 51)R: 5′- TTC CCG AAC TTG ATT CTG AG-3′ (SEQ ID NO: 52) Pax6F: 5′- AGT GGG TTT GAA AAG GGA AC-3′ (SEQ ID NO: 53)R: 5′- ATT GGT GAT GGC TCA AGT GT-3′ (SEQ ID NO: 54) POU5F1F: 5′- GGA CCA GTG TCC TTT CCT CT-3′ (SEQ ID NO: 55)R: 5′- CCA GGT TTT CTT TCC CTA GC-3′ (SEQ ID NO: 56) CD45F: 5′- CCT GCT CAG AAT GGA CAA GT-3′ (SEQ ID NO: 57)R: 5′- TCA GAA CCT TCA GCC TGT TC-3′ (SEQ ID NO: 58) Sox2F: 5′-GAA ATG GGA GGG GTG CAA AA-3′ (SEQ ID NO: 59)R: 5′- ATC GCG GTT TTT GCG TGA GT-3′ (SEQ ID NO: 60)

EXAMPLE 2

Assays were carried out to demonstrate that SB-3 are stem cells capableof differentiating into different cells lineages.

Briefly, SB-3 cells were obtained from a subject in the manner describedabove. SB-3 cells were then cultured in a differentiation medium. Allprimers used for detecting differentiation markers with real time RT-PCRare listed in Table 2 below.

SB-3 cells were induced to express nestin, an early marker for formationof neuron (ectoderm) and islet cells (endoderm). Briefly, SB-3 cellswere cultured in an induction medium that contains 10 nM glucocorticoidand 10% FBS. After 1-month treatment, RNA was extracted and geneexpression was determined by Real Time PCR. Expression of nestin wasdetected.

TABLE 2 GABAR F: 5′-TTATCTCACCCCTTCCTTGG-3′ (SEQ ID NO: 61)R: 5′- GCCATCATGTAGCATTCCTG-3′ (SEQ ID NO: 62) AlbuminF: 5′- TGTGAAACACAAGCCCAAGGCA-3′ (SEQ ID NO: 63)R: 5′-CCCTCCTCGGCAAAGCAGGT-3′ (SEQ ID NO: 64) CD31F: 5′- CAGGCTTCGGCTCAGGCACC-3′ (SEQ ID NO: 65)R: 5′- ATCGGGGCCGGGTGACTTCA-3′ (SEQ ID NO: 66) NR4A2F: 5′-GCTCAAGGAACCCAAGAGAG -3′ (SEQ ID NO: 67)R: 5′-GGCACCAAGTCTTCCAATTT-3′ (SEQ ID NO: 68) MAP-2F: 5′-CGCACACCAGGCACTCCTGG-3′ (SEQ ID NO: 69)R: 5′- CACCTGGCCTGTGGCGGATG-3′ (SEQ ID NO: 70) N-CamF: 5′- CTCCAGCACAGCCCAGGTGC-3′ (SEQ ID NO: 71)R: 5′-TGCTGGCTTCCTTGGCATCATGC-3′ (SEQ ID NO: 72) TauF: 5′-AAGATCGGCTCCACTGAGAA-3′ (SEQ ID NO: 73)R: 5′-GGACGTGGGTGATATTGTCC-3′ (SEQ ID NO: 74) InsulinF: 5′-AGCCTTTGTGAACCAACACC-3′ (SEQ ID NO: 75)R: 5′-GCTGGTAGAGGGAGCAGATG-3′ (SEQ ID NO: 76) TransferrinF: 5′-GAGGCCACTAAGTGCCAGAG-3′ (SEQ ID NO: 77)R: 5′-TTCTTCACCACAGCAACAGC-3′ (SEQ ID NO: 78) TyrosineF: 5′-GCTCAGGAGCTATGCCTCAC-3′ Hydroxylase (SEQ ID NO: 79)R: 5′-ACCTAGCCAATGGCACTCAG-3′ (SEQ ID NO: 80) Neurofilament-F: 5′-AAGTCAGACCAAGCCGAAGA-3′ M (SEQ ID NO: 81)R: 5′-GCACAGGAGACTTGCCTTTC-3′ (SEQ ID NO: 82) Myosin heavyF: 5′-GCTGGAGTCCTCACAGAAGG-3′ chain alpha 6 (SEQ ID NO: 83)(cardiomyocyte) R: 5′-TCTCCAGCTCATGCACATTC-3′ (SEQ ID NO: 84)Myosin light F: 5′-TTCAGTGCTGACCAGATTGC-3′ chain 1 fast (SEQ ID NO: 85)(skeletal R: 5′-AAATGGCTTGCATCATAGGC-3′ myocyte) (SEQ ID NO: 86)Osteocalcin F: 5′-TGAGAGCCCTCACACTCCTC-3′ (OC) (SEQ ID NO: 87)R: 5′-TCAGCCAACTCGTCACAGTC-3′ (SEQ ID NO: 88) a-fetoproteinF: 5′- AAATGCGTTTCTCGTTGCTT-3′ (SEQ ID NO: 1)R: 5′- GCCACAGGCCAATAGTTTGT-3′ (SEQ ID NO: 2) HNF4aF: 5′- TGTGAGTGGCCCCGACCCTG-3′ (SEQ ID NO: 41)R: 5′- ACGATTGTGGCGACGGCTCC-3′ (SEQ ID NO: 42) CD133F: 5′- AGC GAT CAA GGA GAC CAA AG-3′ (SEQ ID NO: 11)R: 5′- AAG CAC AGA GGG TCA TTG AG-3′ (SEQ ID NO: 12) CD44F: 5′-TCGAAGAAGGTGTGGGCAGAAGA -3′ (SEQ ID NO: 27)R: 5′-ATTTCCTGAGACTTGCTGGCCTCT -3′ (SEQ ID NO: 28) CD10F: 5′- GGT TGG GAG CTG ATG AAA CT-3′ (SEQ ID NO: 5)R: 5′- GAA TAG GGC TGG AAC AAG GA-3′ (SEQ ID NO: 6) CXCR4F: 5′- GTT GGC TGA AAA GGT GGT CT-3′ (SEQ ID NO: 35)R: 5′- CAC AAC CAC CCA CAA GTC AT-3′ (SEQ ID NO: 36) NestinF: 5′- TGCCCGGCACTGGGGACTTA-3′ (SEQ ID NO: 49)R: 5′- TAGCGGGCCAGGCCTCTCAG-3′ (SEQ ID NO: 50) CD105F: 5′- CAC TAG CCA GGT CTC GAA GG-3′ (SEQ ID NO: 7)R: 5′- CTG AGG ACC AGA AGC ACC TC -3′ (SEQ ID NO: 8)

Endoderm cells are characterized by their polygonal shapes. Expressionof two hepatocyte markers (transferrin and albumin) and three islet cellmarkers (insulin, alpha-Fetoprotein, and HNF4 alpha) were detected. Inaddition, both Western blot and ELISA also detected expression ofalbumin in differentiated cells. These results indicate that SB-3 cellswere differentiated into hepatocytes and some were differentiated intoislet cells.

Ectoderm cells are characterized by their filament-like feature.Differentiation of SB-3 cells to neuronal cells were confirmed by realtime RT-PCR, which detected expression of many neuronal markers,including CD133, nestin, microtubule-associate protein II, GABAreceptor, NR4A2, N-cam, tyrosine hydroxylase, neurofilament, and Tau.

Further, SB-3 cells were induced to differentiate into adipocytes orosteogenic cells, i.e., mesoderm cells. SB-3 cells were cultured inmedia A, B, C, D, and E sequentially. Then, the medium was replaced byan adipocyte differentiation medium (Invitrogen) for 8 weeks. Theadipocytes were stained using Oil-red-O and detected in an OD490 ELISAspectrophotometer. Alternatively, the medium was replaced by anosteogenesis medium (Invitrogen). Osteogenic cells were observed 2-4weeks after the medium replacement. Osteogenic cells were stained withAlizarin Red, and detected by extracting from the cells Alizarin Red,which was measured at OD 405 nm in a spectrophotometer. The results showthat SB-3 cells can be differentiated to adipocytes or osteogenic cells.

For induction to other mesoderm cells, SB-3 cells were cultured in themedium that contained 10 nM glucocorticoid and 10% FBS. After 1-monthtreatment, RNA was extracted from the cells, and expression of severalgenes was determined by Real Time PCR. Detectable expression of myosinheavy chain and skeletal myosin light chain indicates SB-3 cells weredifferentiated to cardiomyocyte and skeletal muscle cells.

The above results suggest that, upon receipt of a signal, SB-3 cells areactivated and differentiate into suitable tissues to repair the damagedtissues. Thus, these cells contain adult pluripotent stem cells and canbe used for gene therapy, gene banking, drug screening, and creatinguniversal donor cells. Also, these cells could be used to treatdegenerative diseases, autoimmune diseases, or cancer.

EXAMPLE 3

SB-4 cells were cultured in four different types of media and analyzedto confirm that differentiation was successful. To test mesodermdifferentiation, cells were cultured in adipogenesis and osteogenesismedia. Oil-Red O and Alizarin Red staining indicated significantincreases in adipocytes and osteocytes, respectively, when compared totheir negative controls.

To investigate ectoderm differentiation ability, SB-4 cells werecultured in neuron differentiation medium. Results from ICCneurofilament staining and real time-PCR confirmed that SB-4 has neurondifferentiation ability.

SB-4 cells were also induced to differentiate to hepatocytes byculturing them successively in three different media as follows.

First, SB-4 cells were cultured for five days in a DMEM/high glucosemedium containing 3% horse serum, lx antimycotic, 1×L-glutamine, and 5ng/mL activin. Next, the cells were further cultured for fifteen days inthe same medium except that activin was replaced with 20 ng/mL bFGF and5 ng/mL BMP2. Finally, the cells were cultured in a Hepato ZYME-SFMmedium containing 1% horse serum, 10 ng/mL HGF, 10 nM glucocorticoidDEX, and 10 ng/mL OSM for fifteen days or until hepatocyte-like cellsappear. The culturing medium was routinely refreshed at least twice aweek. The hepatocyte-like cells were observed by microscope. Further,they were tested for expression of heptocyte markers by real time RT-PCRwith the primers listed in Table 3 below:

TABLE 3 Albumin F: 5′-GAAACATTCACCTTCCATGC-3′ (SEQ ID NO: 89)R: 5′-ACAAAAGCTGCGAAATCATC-3′ (SEQ ID NO: 90) TransferrinF: 5′-GGAGCCTTCAAGTGTCTGAA-3′ (SEQ ID NO: 91)R: 5′-GTTGAGAAGCTCCCAGATCA-3′ (SEQ ID NO: 92) HNF3BF: 5′-CCATGCACTCGGCTTCCAGTATG-3′ (SEQ ID NO: 93)R: 5′-CGCCGACATGCTCATGTACGTG-3′ (SEQ ID NO: 94)

The results show that the hepatocyte-like cells indeed expressed threeheptocyte markers, i.e., albumin, transferrin, and HNF-3beta.

We believe that these hepatocyte-like cells can have the ability tometabolize and successfully remove toxins, such as ammonia, andsynthesize urea for the detoxification. Thus, the hepatocyte-like cellscan be used as the artificial liver system which is able to replicatehuman liver functions and support patients with acute liver failure. Inaddition, our hepatocyte-like cells can produce albumin which is animportant product for marketing since it can be used as a carrier forsmall pill/drug.

Thus, these hepatocyte-like cells can be used to make artificial liversystem and facilitate drug delivery. These cells would also be usefulfor treating liver degenerative disease and liver cancer.

Other Embodiments

All of the features disclosed in this specification may be combined inany combination. Each feature disclosed in this specification may bereplaced by an alternative feature serving the same, equivalent, orsimilar purpose. Thus, unless expressly stated otherwise, each featuredisclosed is only an example of a generic series of equivalent orsimilar features.

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, other embodiments are within the scope of the followingclaims.

What is claimed is:
 1. A method for treating a muscle injury or amuscle-degenerative disease, the method comprising administering to asubject in need thereof an effective amount of isolated somatic stemcells prepared by a procedure including: incubating a bodily fluidsample containing a plurality of cells with EDTA or heparin in acontainer until the sample is separated into an upper layer and a lowerlayer, collecting the upper layer, isolating from the upper layer apopulation of small somatic stem cells that are 0.3-6.0 micrometers insize, and culturing the small somatic stem cells in a medium containingR-Spondin-1, SCF, G-CSF, bFGF, EGF, and PDGF, whereby the isolatedsomatic stem cells are prepared.
 2. The method of claim 1, wherein thesomatic stem cells are CD10+, CXCR4+, and CD31+.
 3. The method of claim1, wherein the somatic stem cells are CD105+, CD44+, and Nestin+.
 4. Themethod of claim 1, wherein the bodily fluid sample is a blood sample ora bone marrow sample.
 5. An extracorporeal bioartificial liver devicecomprising a cartridge that contains an array of hollow fibers and livercells placed in the extracapillary space between the hollow fibers,wherein the hollow fibers are each formed of a membrane having a poresize of 0.1 μm to 0.3 μm, wherein the liver cells are prepared by aprocedure including: incubating a bodily fluid sample containing aplurality of cells with EDTA or heparin in a container until the sampleis separated into an upper layer and a lower layer, collecting the upperlayer, isolating from the upper layer a population of small somatic stemcells that are 0.3-6.0 micrometers in size, culturing the small somaticstem cells in a medium containing R-Spondin-1, SCF, G-CSF, bFGF, EGF,and PDGF, whereby isolated somatic stem cells are prepared, culturingthe isolated somatic stem cells in a first differentiating mediumcontaining activing, culturing the isolated somatic stem cell in asecond differentiating medium containing basic FGF and BMP2, culturingthe isolated somatic stem cell in a third differentiating mediumcontaining HGF, and DEX, and OSM, and collecting liver cells thusobtained, the liver cells expressing albumin, transferrin, and HNF3B. 6.The device of claim 5, wherein the cartridge is in a cylindrical shapehaving a first opening at one terminus and a second opening on the otherterminus, and wherein the first opening is affixed to a first passageand the second opening is affixed to a second passage, the two passagesextending away from the cartridge.
 7. A method of treating acute liverfailure, the method comprising: identifying a subject in need oftreatment, attaching the device of claim 5 to an artery of the subjectthrough the first passage and a vein of the subject through the secondpassage, perfusing blood from the subject through the capillary spaceinside each of the hollow fibers in the cartridge, and allowingcleansing of blood by permitting the crossover of toxic solutes from theblood to the liver cells cultured in the extracapillary space betweenthe hollow fibers and also allowing the diffusion of vital metabolitesfrom the liver cells to the blood returning to the subject undergoingtreatment.
 8. A method of making an extracorporeal bioartificial liverdevice, comprising: incubating a bodily fluid sample containing aplurality of cells with EDTA or heparin in a container until the sampleis separated into an upper layer and a lower layer, collecting the upperlayer, isolating from the upper layer a population of small somatic stemcells that are 0.3-6.0 micrometers in size, culturing the small somaticstem cells in a medium containing R-Spondin-1, SCF, G-CSF, bFGF, EGF,and PDGF, whereby isolated somatic stem cells are prepared, culturingthe isolated somatic stem cells in a first differentiating mediumcontaining activing, culturing the isolated somatic stem cell in asecond differentiating medium containing basic FGF and BMP2, culturingthe isolated somatic stem cell in a third differentiating mediumcontaining HGF, and DEX, and OSM, collecting liver cells thus obtained,the liver cells expressing albumin, transferrin, and HNF3B, and placingthe liver cells within the extracapillary space between hollow fibers ina cartridge, wherein the hollow fibers are each formed of a membranehaving a pore size of 0.1 μm to 0.3 μm.
 9. The method of claim 8,wherein the cartridge is in a cylindrical shape having a first openingat one terminus and a second opening on the other terminus, and whereinthe first opening is affixed to a first passage and the second openingis affixed to a second passage, the two passages extending away from thecartridge.